Aging of the cardiovascular system is characterized by a reduction in cardiac function, and cardiovascular disease frequently strikes in the latter half of life. Many experimental findings suggest that adult mammalian myocardium has a population of resident cardiac stem cells with differentiation potential for cardiomyocytes and other cell types like endothelial cells and vascular smooth muscle cells. Like other stem cells, cardiac stem cells may be subject to senescent changes with increasing age, possibly reducing their regenerative potential. Our laboratory has developed a genetic cell fate-mapping approach to quantify cardiomyocyte turnover, and we have used this system to demonstrate that in the first half of life, cardiomyocytes are not replaced by stem/precursor cells in the absence of injury; however, after myocardial infarction, there is activation of a stem/precursor pool. Genetic fate-mapping is a powerful approach for cell tracking, but it cannot simultaneously quantify cell division rates of the identified cell populations. Until recently, methods for tracking cell division relied mainly on BrdU incorporation or tritium (3H) labeling; these techniques have limitations because they do not always have sufficient resolution to track cell divisions quantitatively, and they can be toxic under certain circumstances. A new technology called Multi-Isotope Imaging Mass Spectrometry (MIMS) has the potential to overcome these drawbacks, permitting quantitative assessment of cell division history. The sensitivity of MIMS allows changes to be monitored over a wide range of times because a labeling pulse is generated using stable, nonradioactive isotopes that are non-toxic for animals and humans. MIMS can monitor cell division in vitro and in vivo with time scales potentially ranging from minutes to years, since stable isotopes do not decay or emit radiation. In this proposal, we describe experiments to study myocardial progenitor recruitment in aging mice using the complementary techniques of genetic cell fate-mapping and MIMS, which in combination will facilitate quantitative tracking of different cell populations and their relative rates of cell division. Our specific aims are: Aim 1. To test the hypothesis that in the absence of injury, cardiomyocytes are not significantly refreshed by precursor cells in the aging mouse. Aim 2. To test the hypothesis that aging reduces the capacity of mammalian myocardium to refresh cardiomyocytes by the stem/precursor cell pool following injuries representative of human diseases. Aim 3: To test the hypothesis that stable isotope MIMS quantification combined with genetic fate mapping reveals a low rate of basal cardiomyocyte cell division with no measurable contribution of cell division from a precursor pool in adult mammalian myocardium during aging. Aim 4: To test the hypothesis that, following injury, cardiomyocytes are replenished primarily by stem cells and not by cell division of pre-existing cardiomyocytes irrespective of age. PUBLIC HEALTH RELEVANCE: Heart disease frequently strikes in the latter half of life, and healing and regeneration of heart tissue may be impaired by the aging process. This project uses new sophisticated methods to determine if the healing process of the heart changes as mice age. These experiments will provide new insight into the aging process and the most common causes of death in the United States.